The eukaryotic ribonucleoprotein reverse transcriptase telomerase elongates chromosome 3' ends de novo, adding back telomeric simple-sequence repeats that are lost with each cycle of genome replication due to incomplete end-replication by the conventional DNA synthesis machinery. Down- regulation of telomerase after early embryogenesis sets an upper limit for human somatic tissue renewal, which is encountered prematurely in human telomerase deficiencies or telomeropathies including a bone marrow failure syndrome, pulmonary fibrosis and other disorders. On the other hand, tumor cells over-activate telomerase to support their indefinite proliferative capacity. Telomerase is unique among polymerases in its reiterative copying of a template within the enzyme's integral RNA subunit. Instead of generating product that is RNA-DNA duplex, telomerase releases single-stranded telomeric repeat DNA. The elaborate catalytic cycle required to support this activity arises from collaboration of telomerase reverse transcriptase (TERT), telomerase RNA (TER) and the numerous other subunits of a biologically active telomerase holoenzyme. The long-term objective of research funded by this RO1 is to determine components, structures, biochemical mechanisms and cellular regulations of telomerase. These goals will inform fundamental knowledge about mechanisms of genome synthesis, genome stability, control of cellular proliferation and tumorigenesis and also specificity principles for dynamic protein-nucleic acid interaction. The Specific Aims build from and extend the Collins lab two-decade track record of insights about telomerase composition, assembly, activity, recruitment to telomeres and regulation using two enabling cellular systems studied in parallel: human cells and the ciliate Tetrahymena. In the current funding period we accomplished the first holoenzyme structure determination with resolution sufficient to place all of the subunits and derive a model of TER tertiary structure. We discovered unanticipated mechanisms underlying several steps of the telomerase catalytic cycle of repeat synthesis in vitro and subunit interactions that mediate telomerase recruitment to telomeres in vivo. We will exploit these advances and our single-molecule-level analysis of human telomerase architecture to determine structures of the human telomerase holoenzyme as well as atomic-resolution structures of Tetrahymena telomerase holoenzyme proteins (Aim 1), define the biochemical and molecular basis for the specificities of telomerase-DNA interaction (Aim 2) and test hypotheses about how telomerase finds an extreme chromosome 3' terminus and elongates it in coordinates with other DNA replication machinery (Aim 3). These studies inform strategies of telomerase modulation for boosting cellular regeneration in human tissues and cell transplantations, and provide targets for anti-cancer therapy.